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1 Mount Holyoke College, South Hadley, MA
2 Hospital for Sick Children, Toronto, Canada
3 Friedrich Miescher Institute, Basel, Switzerland
4 University of Barcelona, Barcelona, Spain
* Corresponding author: stepsupo{at}mtholyoke.edu
When two sponge tissue fragments from the same individual are adjoined, isogeneic recognition occurs, and the fragments fuse. On the other hand, if the two pieces are from different individuals, allogeneic recognition occurs, followed by failure of fusion and, presumably, death of cells at the graft contact zone (1, 2). It has been proposed that two cell types, archaeocytes and gray cells, are involved in sponge allogeneic recognition. Archaeocytes are large phagocytic cells with large nucleoli; they are commonly found in wound healing and regenerating areas of the sponge (1). Gray cells are motile cells that contain many dense cytoplasmic granules (1); they do not have well-defined pseudopodia and are commonly found in growing regions of the sponge but are rare in the marginal region (3).
Apoptosis (programmed cell death) in sponges was first demonstrated by TUNEL nick-end labeling assay in hibernating sponges that had undergone tissue regression as a naturally occurring winter event (4). The development of an alternate method for detecting apoptosis was the finding that a series of caspases (proteases) delineated sequential enzymes leading to cell death (5). Of these, caspase-3 enzyme was the final and most important protease. From this have emerged antibodies for the detection of caspase-3 (6). Biochemical methods were used by Wiens et al. (7) to study apoptotic events in extracts of the marine sponge Geodia cydonium, where caspase-3 activity was found to be greatly increased in allograft extracts when compared with isografts. The objectives of the present research are (a) to determine by immunohistochemistry whether cell death at the allograft contact zone is a result of apoptosis rather than necrosis (death by injury that may result from mechanical damage to the cells) and (b) to identify the cell type or types, if any, that undergo apoptosis at the contact zone.
To examine whether the death of cells at the contact zone is a result of apoptosis, we used an indirect labeling technique that provides precise tissue localization of active caspase-3 as a marker for cells undergoing apoptosis. Processed cells that were reactive were visualized by the presence of a dark brown precipitate when color was developed with diaminobenzidine (DAB).
Individuals of Microciona prolifera were collected by the Marine Resources Center of the Marine Biological Laboratory (Woods Hole, MA) and were maintained until use in a tank with running cold seawater. The two sponge pieces for isografts and allografts were held together with number zero stainless steel pins attached to a Styrofoam board floating in a tank of running cold seawater. The grafts were fixed at 6-h intervals from 0 to 24 h in 3.7% formaldehyde in MBL artificial seawater (MBLASW) overnight, then washed and dehydrated in a series of ethyl alcohol concentrations from 30% to 70% in MBLASW. Spicules were dissolved by overnight treatment of grafts with 4% hydrofluoric acid in 70% ethanol. The grafts were then embedded in paraffin and sectioned at 7 µm.
The slides of sectioned isografts and allografts were deparaffinized, hydrated, and treated with 3% hydrogen peroxide to remove endogenous peroxidase activity. Tissue presumed to possess caspase-3 activity was incubated using purified rabbit IgG anti-active human caspase-3, which had been generated by affinity purification from a caspase-3 enzyme fragment (Pharmingen #559565); the antibody was used at a concentration of 0.25 µg/ml for 2 h at room temperature. The slides were then washed with phosphate-buffered saline (PBS, pH 7.5) (Sigma), followed by treatment with appropriate biotinylated secondary antibody (Vector Laboratories). After another wash in PBS, the slides were treated with avidin-biotinylated enzyme complex (VectaStain Elite ABC Kit, Vector Laboratories) before the peroxidase substrate DAB (Sigma) was applied. The slides were washed, dehydrated, mounted, and photographed with a digital camera on a Zeiss microscope.
Isograft sections treated with anti-active caspase-3 primary antibody did not stain, indicating that apoptosis did not occur in either the line of contact or any of the cells in the grafts during a 24-h period (Fig. 1A). On the contrary, treatment of allograft sections with the same primary antibody showed that cell death at the contact zone was indeed a result of apoptosis. Cells near the contact zone started to undergo apoptosis 6 h after grafting (data not shown). The line of contact became most apparent at 24 h (Fig. 1B). The longer the period of allogeneic contact, the higher the number of large, elongated apoptotic cells accumulated at the contact zone. The morphology of these cells was consistent with their identity as archaeocytes.
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We conclude that cell death at the allograft contact zone is a result of apoptosis rather than tissue necrosis, and that the apoptotic cells are most likely archaeocytes, not gray cells.
The ability to recognize self from non-self has been conserved throughout evolution. A better knowledge of this process in the sponge model should enhance our understanding of allogeneic recognition and its regulation in more complex species, including humans. The knowledge gained from such basic mechanisms should be of use in developing effective specific drugs that can abrogate the pathological effects of allogeneic responses (8).
Acknowledgments
Mount Holyoke College HHMI Cascade Mentoring Program; Research Experience for Undergraduates of the Boston University Marine Program; Friedrich Miescher Institute of the Novartis Research Foundation. X. F.-B. holds a Ramón y Cajal tenure-track position from the Ministerio de Ciencia y Tecnología (MCyT), Spain, and acknowledges the support of grant BIO2002-00128 from the MCyT.
Literature Cited
This article has been cited by other articles:
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  C. Sabella, E. Faszewski, L. Himic, K. M. Colpitts, J. Kaltenbach, M. M. Burger, and X. Fernandez-Busquets Cyclosporin A Suspends Transplantation Reactions in the Marine Sponge Microciona prolifera J. Immunol., November 1, 2007; 179(9): 5927 - 5935. [Abstract] [Full Text] [PDF]
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